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Identification and Transcriptional Control of Caulobacter crescentus Genes Encoding Proteins Containing a Cold Shock Domain. Elza A. S. Lang, 2004.The cold shock proteins are small peptides that share a conserved domain, called the cold shock domain (CSD), that is important for nucleic acid binding . The Caulobacter crescentus genome has four csp genes that encode proteins containing CSDs . Three of these (cspA, cspB, and cspC) encode peptides of about 7 kDa and are very similar to the cold shock proteins of other bacteria . Analysis by reverse transcription-PCR of the fourth gene (cspD), which was previously annotated as encoding a 7-kDa protein, revealed that the mRNA is larger and probably encodes a putative 21-kDa protein, containing two CSDs . A search in protein sequences databases revealed that this new domain arrangement has thus far only been found among deduced peptides of  -proteobacteria .
Expression of each Caulobacter csp gene was studied both in
response to cold shock and to growth phase, and we have found
that only cspA and cspB are induced by cold shock, whereas cspC
and cspD are induced at stationary phase, with different induction
rates . The transcription start sites were determined for each
gene, and a deletion mapping of the cspD promoter region defined
a sequence required for maximal levels of expression, indicating
that regulation of this gene occurs at the transcriptional level .
Deletion of cspA, but not cspD, caused a reduction in
viability when cells were incubated at 10°C for prolonged times,
suggesting that cspA is important for adaptation to a low
temperature .
Maternal-Fetal Transfer and Amniotic Fluid Accumulation of Nucleoside Analogue Reverse Transcriptase Inhibitors in Human Immunodeficiency Virus-Infected Pregnant Women. Hélène Chappuy, 2004.This study was performed to investigate placental transfer of nucleoside analogue reverse transcriptase inhibitors (NRTIs) and their concentrations in amniotic fluid when given to human immunodeficiency virus (HIV)-infected pregnant women . A total of 100 HIV type 1-infected mothers receiving antiretroviral therapy, including one or more NRTIs, for clinical indications at the time of delivery were enrolled . Maternal blood samples and amniotic fluid were obtained during delivery or cesarean section, and paired cord blood samples were obtained by venipuncture immediately after delivery . Drug concentrations were measured by using high-performance liquid chromatography . A significant relationship between concentrations in maternal and cord plasma samples was found for zidovudine, lamivudine, stavudine, and didanosine . The ratio between the concentrations in cord and maternal plasma samples (R) was high for zidovudine (R = 1.22), its glucuronide metabolite (3'-azido-3'-deoxythymidine-ß-D-glucuronide) (R = 1.01), stavudine (R = 1.32), lamivudine (R = 0.93), and abacavir (R = 1.03) and was low for didanosine (R = 0.38) . The ratio between the concentrations in amniotic fluid and cord plasma samples was high for zidovudine (R = 2.24), its glucuronide metabolite (R = 2.83), stavudine (R = 4.87), and lamivudine (R = 3.99) and was lower for didanosine (R = 1.14) . These findings indicate that most NRTIs cross the placenta by simple diffusion and are concentrated in the amniotic fluid, probably through fetal urinary excretion . The efficacy or toxicity of NRTIs may vary according to placental transfer . Stable-Isotope Probing of Microorganisms Thriving at Thermodynamic Limits: Syntrophic Propionate Oxidation in Flooded Soil. Tillmann Lueders, 2004.Propionate is an important intermediate of the degradation of organic matter in many anoxic environments . In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H2-consuming methanogens . We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP) . After 7 weeks of incubation with [13C]propionate (<10 mM) and the oxidation of  30 µmol of 13C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were 13C labeled to a larger extent than those of the bacterial partners . Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp . to predominate in "heavy" 13C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation . Among the Archaea, mostly Methanobacterium and Methanosarcina spp . and also members of the yet-uncultured "rice cluster I" lineage had incorporated substantial amounts of 13C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [13C]acetate released by the syntrophs . With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP . This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms .
Detection and Quantification of the Red Tide Dinoflagellate Karenia brevis by Real-Time Nucleic Acid Sequence-Based Amplification. Erica T. Casper, 2004.Nucleic acid sequence-based amplification (NASBA) is an isothermal method of RNA amplification that has been previously used in clinical diagnostic testing . A real-time NASBA assay has been developed for the detection of rbcL mRNA from the red tide dinoflagellate Karenia brevis . This assay is sensitive to one K . brevis cell and 1.0 fg of in vitro transcript, with occasional detection of lower concentrations of transcript . The assay did not detect rbcL mRNA from a wide range of nontarget organisms and environmental clones, while 10 strains (all tested) of K . brevis were detected . By the use of standard curves based on time to positivity, concentrations of K . brevis in environmental samples were predicted by NASBA and classified into different levels of blooms per the Florida Fish and Wildlife Conservation Commission (FWC) system . NASBA classification matched FWC classification (based on cell counts) 72% of the time . Those samples that did not match were off by only one class . NASBA is sensitive, rapid, and effective and may be used as an additional or alternative method to detect and quantify K . brevis in the marine environment . Analysis of ftsQ Mutant Alleles in Escherichia coli: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins. Joseph C. Chen, 2002.FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli . To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature . Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting . The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature . FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum . These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery . Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities . Cell Death in Pseudomonas aeruginosa Biofilm Development. Jeremy S. Webb, 2003.Bacteria growing in biofilms often develop multicellular, three-dimensional structures known as microcolonies . Complex differentiation within biofilms of Pseudomonas aeruginosa occurs, leading to the creation of voids inside microcolonies and to the dispersal of cells from within these voids . However, key developmental processes regulating these events are poorly understood . A normal component of multicellular development is cell death . Here we report that a repeatable pattern of cell death and lysis occurs in biofilms of P . aeruginosa during the normal course of development . Cell death occurred with temporal and spatial organization within biofilms, inside microcolonies, when the biofilms were allowed to develop in continuous-culture flow cells . A subpopulation of viable cells was always observed in these regions . During the onset of biofilm killing and during biofilm development thereafter, a bacteriophage capable of superinfecting and lysing the P . aeruginosa parent strain was detected in the fluid effluent from the biofilm . The bacteriophage implicated in biofilm killing was closely related to the filamentous phage Pf1 and existed as a prophage within the genome of P . aeruginosa . We propose that prophage-mediated cell death is an important mechanism of differentiation inside microcolonies that facilitates dispersal of a subpopulation of surviving cells . Chemical and Biological Interactions during Nitrate and Goethite Reduction by Shewanella putrefaciens 200. D. Craig Cooper, 2003.Although previous research has demonstrated that NO3- inhibits microbial Fe(III) reduction in laboratory cultures and natural sediments, the mechanisms of this inhibition have not been fully studied in an environmentally relevant medium that utilizes solid-phase, iron oxide minerals as a Fe(III) source . To study the dynamics of Fe and NO3- biogeochemistry when ferric (hydr)oxides are used as the Fe(III) source, Shewanella putrefaciens 200 was incubated under anoxic conditions in a low-ionic-strength, artificial groundwater medium with various amounts of NO3- and synthetic, high-surface-area goethite . Results showed that the presence of NO3- inhibited microbial goethite reduction more severely than it inhibited microbial reduction of the aqueous or microcrystalline sources of Fe(III) used in other studies . More interestingly, the presence of goethite also resulted in a twofold decrease in the rate of NO3- reduction, a 10-fold decrease in the rate of NO2- reduction, and a 20-fold increase in the amounts of N2O produced . Nitrogen stable isotope experiments that utilized  15N values of N2O to distinguish between chemical and biological reduction of NO2- revealed that the N2O produced during NO2- or NO3- reduction in the presence of goethite was primarily of abiotic origin . These results indicate that concomitant microbial Fe(III) and NO3- reduction produces NO2- and Fe(II), which then abiotically react to reduce NO2- to N2O with the subsequent oxidation of Fe(II) to Fe(III) .
The High Salt Requirement of the Moderate Halophile Chromohalobacter salexigens DSM3043 Can Be Met Not Only by NaCl but by Other Ions. Kathleen O'Connor, 2003.The growth rate of Chromohalobacter salexigens DSM 3043 can be stimulated in media containing 0.3 M NaCl by a 0.7 M concentration of other salts of Na+, K+, Rb+, or NH4+, Cl-, Br-, NO3-, or SO42- ions . To our knowledge, growth rate stimulation by a general high ion concentration has not been reported for any organism previously .
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